The present study shows that Gluc expressing VRPs represent a useful tool to determine neutralizing antibodies without the need of infectious CHIKV particles. VRPs were produced by cotransfection of a CHIKV replicon expressing Gluc and two helper RNAs. First studies describing the production of VRPs involved the cotransfection of a single helper RNA encoding all structural proteins [3, 31]. However, in this constellation a single RNA recombination event is sufficient to reproduce a full-length genome resulting in production of infectious particles . Expressing the structural proteins via two helper RNAs circumvents this problem and it was shown that using this strategy production of infectious particles due to recombination is negligible [2, 6], which is an important aspect with regard to biosafety issues.
For certain alphaviruses, like SFV and SINV, expression of the structural proteins was described to be dependent on an enhancer sequence located in the 5’ terminal part of the sequence encoding the capsid protein [32, 33]. To ensure in these cases efficient translation of the envelope proteins in a bipartite helper packaging system, the enhancing sequence of the capsid protein was fused 5’-terminally to the envelope genes [6, 7]. However, for VEEV a split helper system was described in which the envelope proteins were expressed without a capsid translation enhancer . The latter implicates that the enhancer sequence is not necessarily needed in the VEEV context . This seems to be also the case for CHIKV, as no stable hairpin structure comparable with capsid enhancers of SFV and SINV could be predicted in the corresponding region of the CHIKV genome. Nevertheless, it was also observed that the CHIKV replicon could be efficiently packed using split helper RNAs of SFV (A. Merits and A. Lulla, unpublished data). Therefore the effect of classical SFV capsid enhancer was tested also in the context of CHIKV helper RNAs. In this experiment the presence of enhancer sequence not only failed to increase but reduced the VRP production (A. Lulla, V. Lulla, unpublished data). Therefore in our split helper system the helper-E construct used did not contain any capsid enhancer and still allowed efficient production of VRPs.
Alphaviruses are known to be transmitted by insect vectors, which exhibit body temperatures below 37°C. Hence, the VRP production was also tested at lower temperature and has proven to be more efficient at 32°C than at 37°C. Reducing the temperature to 28°C even slightly increased the VRP yield further but simultaneously slowed down the production process resulting in a prolonged production time (data not shown).
To facilitate and accelerate the readout of the NT assay we established a VRP system expressing Gluc, which is secreted into the supernatant . Hence, measuring can directly be performed from the supernatant without the need of lysing cells. The latter was described to be necessary for the pseudotyped lentiviral vector-based CHIKV NT assay, which used Fluc as reporter protein . In addition, the humanized Gluc has been described to be 1000-fold more sensitive compared to humanized Rluc or Fluc [28, 34]. Furthermore, Gluc is very stable (half-life around 6 days ), which allows storage of the supernatant at 4°C for several days without significantly loosing activity . On the other hand, due to the high sensitivity, Gluc reporter assays are likely to be prone to pipetting errors . Hence, pipetting smaller volumes when working in the 96-well format at lower MOI might be one reason for the higher deviations observed in this experimental setup. As already described, it is not recommended to use a pipetting volume below 10 μl for Gluc assays . Nevertheless, when using an MOI of 5, reliable results were also obtained in the 96-well format. Using an even higher MOI is not recommended. Besides consuming excessive amounts of VRPs, the risk to exhaust the assay exists. Also, especially at 24 h post infection, the amount of Gluc released into the supernatant after using an MOI of 50 was so high that samples had to be diluted to allow measurement of Gluc activity (data not shown). This could result in an additional source of variation.
The 96-well format might especially be favorable to use when only small sample volumes are available or when neutralizing titers have to be determined for an extensive number of samples. Working in a 96-well format allows to directly transfer the Gluc containing supernatant from the NT assay plate to a corresponding 96-well readout plate using a multichannel pipette thereby avoiding time consuming single pipetting steps. Furthermore, both the recently described pseudotyped lentiviral vector-based NT assay  and our VRP based NT assay have the advantage that the use of infectious CHIKV particles is avoided. However, for the lentiviral system readout is performed several days after transduction, whereas our VRP based assay allows carrying out the NT assay including readout within one day.
Performing different types of NT assays results in fairly different scales of NT titers. This was also observed when comparing the lentiviral vector-based NT assay with a classical plaque neutralization assay . Similarly, the antibody dilutions for which the infectivity was inhibited by 50% were different for each sample comparing our VRP based assay with a plaque neutralization assay. Nevertheless, the order of neutralization potency among the samples was consistent (Table 1) indicating that the assay is suitable to determine comparative neutralization activities. Furthermore, the fact that no infectious CHIKV particles are needed and that readout can already be performed after 6 h makes the established Gluc VRP based NT assay a valuable tool to study patient or animal serum samples. Besides diagnostic purposes, analyses of neutralizing antibodies in human sera will help to understand immune mechanisms involved in CHIKV disease and analyses in animals will be useful in evaluation steps during CHIKV vaccine development.